Fibronectin glycation increases IGF-I induced proliferation of human aortic smooth muscle cells

Fibronectin and other routine reagents were obtained from Sigma Chemical Co. (St. Louis, MO), IGF-I and Des (1–3) IGF-I were supplied by GroPep (Adelaide, South Australia), PDGF-BB and all materials for cell culture were obtained from Life Technologies (Gaithersburg, MD). Iodinated [¹²⁵I] des (1–3) IGF-I, [¹²⁵I] labelled Protein A (100 mCi/mL) and ECL detection kit were purchased from Amersham (Arlington Heights, IL). Antibody 1.2, a monoclonal antibody to the C-terminus of the IGF-I receptor, was a kind gift from Dr. Kenneth Siddle (Cambridge, UK). Anti-IGFBP-4 antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-phosphotyrosine antibody (RC20H) conjugated to horseradish peroxidase was purchased from BD Transduction Laboratories (Lexington, KY).

FN (2.5 mg) was incubated in phosphate-buffered saline (PBS) (pH 7.4) with 500 mM D-glucose at 37°C in the presence of protease inhibitors (1.5 mM phenylmethylsulfonyl fluoride [PMSF], 0.5 mM EDTA) and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin) for 6 weeks. Control FN was incubated under the same conditions without glucose. Unreacted sugar was removed by dialysis against PBS. Characteristic fluorescence of the AGE compound 2-(2-furoyl)-4(5)-(2-furanyl)-1 H-imidazole (FFI) was observed at 440 nm after excitation at 375 nm [26]. FN structural modification secondary to advanced glycation was established by assessing bityrosine formation (400 nm upon excitation at 325 nm) and tryptophan quenching (334 nm upon excitation at 275 nm) [27] (Figure 1). Control and AGE-modified FN were evaluated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [28] using a 5% stacking gel and a 7.5% separating gel and they exhibited indistinguishable electrophoretic patterns, without any sign of degradation (data not shown). Control and AGE-FN were plated at 4 μg/cm² either onto 12-well tissue culture plates or onto 75 cm² culture flasks for 1 h at room temperature (RT). The plates were gently rinsed three times with PBS before SMCs were allowed to adhere.

An immortalized SMC line (AALTR-16) was kindly provided by Dr. J.K. McDougall (Fred Hutchinson Cancer Research Center, Seattle, WA): human aortic SMCs were infected with a retro viral vector containing the E6/E7 open reading frame of human papilloma virus type 16. The E6 protein binds and promotes the degradation of the wild-type p53 protein and the E7 protein forms an inactivating complex with the product of the retinoblastoma tumor-suppressor gene. These cells did not show any tumorigenic activity in irradiated nude mice [29] and their characterization by immunocytochemical studies and electronic microscopy demonstrated, respectively, expression of alpha -smooth muscle actin and a synthetic phenotype, featured by a prominent endoplasmic reticulum. Lack of oncogenic transformation was evaluated by serum dependent assay (data not shown). The cells were maintained in medium supplemented with 10% FCS, 1 mM l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified atmosphere of 95% air - 5% CO₂, with medium changes three times a week. Every five days, the cells were harvested with a solution of 0.25% trypsin. To study the effect of AGE-FN on SMC proliferation, cells were seeded at 2 × 10⁴ cells/cm² into 96-well plates coated with either FN or AGE-FN and were cultured in medium supplemented with 10% FCS until sub-confluence. After incubation with serum-free medium for 24-h, cells were incubated for 24 h in serum-free medium (baseline) or in medium containing IGF-I (the concentration of 100 ng/mL was used because a dose–response curve did not show a significant effect of 10, 25 and 50 ng/mL of IGF-I on SMC proliferation [data not shown]). After medium aspiration, fresh medium containing 2 mCi/mL of [methyl-3 H] thymidine (Amersham) was added for additional 4 h. The cells were rinsed twice with ice-cold PBS, twice with ice-cold 5% trichloroacetic acid, and twice with ice-cold 95% ethanol. The cells were lysed in 0.3 mL of 1 N NaOH, neutralized with 0.3 mL of 1 N HCI, and counted in a liquid scintillation counter. The results are expressed as the mean ± SEM of four independent experiments performed in duplicates.

SMC were seeded into 12-well plates coated with FN or AGE-FN and were grown in medium supplemented with 10% FCS until sub-confluence. After incubation with serum-free medium for 24-h, the cells were washed with 1 mL of ligand binding buffer and incubated in 0.5 mL of binding buffer containing 25,000 cpm of radiolabeled material (¹²⁵Des (1–3) IGF-I) and unlabeled competitor (Des (1–3) IGF-I) in the following final concentrations: 0, 0.5, 1, 2, 4, 8, 16, 32 and 100 ng/mL as previously described [30]. Total binding was designated as the quantity of labeled ligand bound under these conditions. Nonspecific binding was defined as the radioligand bound in the presence of a 100-fold molar excess of unlabeled ligand. Specific binding was calculated as the difference between total and nonspecific binding.¹²⁵Des (1–3) IGF-I binding kinetics were calculated using a LIGAND computer program [31]. The results are expressed as means ± SEM of three independent experiments performed in duplicates.

IGF-IR protein levels were determined by immunoblotting membranes containing cell lysates of SMC seeded into 100-mm plates coated with FN or AGE-FN, grown in medium supplemented with 10% FCS until sub-confluence and synchronized by serum starvation for 24-h with antibody 1.2, a monoclonal antibody to the C-terminus of the IGF-IR, as previously described [32]. Three independent experiments were performed. IGF-IR autophosphorylation was analyzed by immunoblotting in cell lysates (75 mg) with an antiphosphotyrosine antibody, as previously described [32]; SMC seed into 100-mm plates coated with FN or AGE-FN were grown in medium supplemented with 10% FCS until sub-confluence and after synchronization for 24-h, cells were incubated either with or without IGF-I (100 ng/mL) for 1 min at 37°C. Three independent experiments were performed.

To study the effect of AGE-modified FN on IGFBP-4 secretion, cells were plated at 3 × 10⁴ cells/cm² into 75 cm² culture flasks coated with FN or AGE-FN, and were grown in medium supplemented with 10% FCS until sub-confluence. After being rinsed twice, they were incubated with serum-free medium supplemented with 1 mM l-glutamine and antibiotics for 24-h. The medium was then replaced with or without addition of PDGF-BB (10 ng/mL) for 24-h. The conditioned medium was collected and concentrated by ultrafiltration in 3 kDa MW cut-off Centripep columns (Amicon Inc., Beverly, MA) and protein was determined by the method described by Bradford [33]. Identification of IGFBP-4 was performed according to the anti-IGFBP-4 antibody manufacturer’s Western Blot (WB) protocol (United Biotech, Inc. Mountain View, CA). Briefly, after gel electrophoresis and transference of proteins, the nitrocellulose membrane was washed twice with water and blocked with phosphate buffered saline (PBS) with 3% non-fat dry milk for 20 min at RT. The membrane was then incubated with a 1:2.000 dilution of antibody anti-human IGFBP-4 (rabbit, polyclonal) in PBS 1% non-fat dry milk at 4°C overnight. This was followed by two washes with water and incubation for 2 h at RT with anti-rabbit immunoglobulin G (peroxidase-linked, Amersham) in PBS 1% non-fat dry milk. The membrane was washed twice with water, once with PBS 0.05% Tween-20 and finally four times with water. A chemiluminescent peroxidase substrate (ECL) was applied for 1 min and the membrane was exposed briefly to autoradiographic film. The densities of the bands were determined by scanning densitometry. Three independent experiments were performed.

To study the effect of AGE-modified FN on IGF-IR and IGFBP-4 mRNA content, total RNA from cells from the previous experiment was prepared using TRIzol® reagent (Invitrogen, Carlsbad, CA) accordingly to standard protocols provided by the manufacturer. RNase protection assay was performed as previously described [34] using the following probes: (I) IGF-IR probe, which is a 379 bp fragment of human IGF-IR cDNA cloned into a pGEM3 vetor [35], (II) IGFBP-4 probe, which is a 505-bp fragment of human IGFBP-4 cDNA cloned into a pBSK + vector [36] and (III) 18S RNA riboprobe, which served as an internal control. Three independent experiments were performed.

As shown in Figure 2, IGF-I elicited an increment significantly higher (P < 0.05) in thymidine incorporation in SMC grown on AGE-FN than in cells grown on FN demonstrating the stimulatory effect of AGE on SMC proliferation in the presence of IGF-I. No differences in thymidine incorporation were observed between SMC coated in FN and AGE-FN in the absence of IGF-I.

In order to investigate if AGE-FN promotes increased SMC proliferation by augmenting IGF-IR number and/or affinity, an IGF-I binding assay was performed with Des(1–3) IGF-I, a truncated form of IGF-I (lacking the N-terminal tripeptide Gly-Pro-Glu) that has much reduced IGFBP affinity compared with natural IGF-I [37]. Thus the use of this analog avoids falsely elevated IGF-IR binding sites, which may occur with any binding of IGF-I radioligand to IGFBPs. IGF-binding curves fitted to one binding site (Figure 3). The receptor numbers per cell were not different between SMC plated on FN and on AGE-FN (1.33 × 10⁴ in both conditions), neither were the relative affinities for IGF-IR (Kd of 6.64 × 10⁻¹¹ and 6.31 × 10⁻¹¹, respectively) (P > 0.05). Also, the evaluation of IGF-IR by Western blot did not show any significant difference between the abundance of this protein on SMC grown on FN and AGE-FN (P > 0.05) (Figure 4). Since changes in IGF-IR number and affinity could not explain increased cell proliferation stimulated by IGF-I in presence of AGE-FN, autophosphorylation of the IGF-IR was examined by Western blot with an anti-phosphotyrosine antibody. IGF-I stimulated autophosphorylation of SMC seeded on both FN and AGE-FN did not consistently differ between the two experimental conditions (P > 0.05) (Figure 5).

To additionally investigate if a decrease in IGFBP-4, a binding protein that inhibits IGF-I mitogenic effects, was involved in the increased SMC proliferation determined by AGE-FN, a Western blotting employing an antibody anti- human IGFBP-4 was performed. As shown in Figure 6, after 24 h of incubation in serum-free media, the content of IGFBP-4 in the conditioned media of SMC grown on AGE-FN was similar to the observed in the conditioned media of cells seeded on FN (P > 0.05), however, treatment with 10 ng/mL of PDGF-BB for 24 h evoked opposite effects on the IGFBP-4 content, augmenting (range from 40–60%) this binding protein in the conditioned media from SMC grown on FN (as observed in cells maintained in plastic substratum [data not shown] and as described in previous studies [22, 24]) and decreasing (range from 28–46%) it in the conditioned media of SMC cultivated in AGE-FN (P < 0.05). This result suggests that the interaction of SMC with AGE-FN modifies the dynamic of IGFBP-4 secretion induced by PDGF-BB, resulting in elevation of local free IGF-I, which may contribute to SMC proliferation.

We next examined the effect of AGE-FN on IGF-IR and IGFBP-4 expression before and after treatment with PDGF-BB, given the known influence of PDGF isoforms on the IGF-I axis in SMC [22, 24]. IGF-I expression was not evaluated because preliminary experiments have demonstrated low expression levels of this growth factor in the studied cell line (data not shown). IGF-IR mRNA and IGFBP-4 mRNA abundances were not affected by AGE-FN in comparison to the abundance observed in cells grown on FN substrata after 24 h, and treatment with PDGF-BB had no effect on the content of IGF-IR and IGFBP-4 mRNAs in relation to untreated cells in both substrata (P > 0.05) (Figure 7), suggesting that the modulation of IGBBP-4 secretion observed after PDGF-BB treatment is probably due to post-transcriptional events.